Enzymes known as transglutaminases are primarily responsible for the stabilisation of many protein aggregates, such as for example, in blood clot formation. The cross-linking of proteins by the action of transglutaminases is the major way in which, for example, fibrin clots are stabilised. In mammals, stabilisation of blood clots is brought about by a transglutaminase, known as Factor XIIIa, which catalyses the formation of cross-linking between the fibres of fibrin. Cross-linked blood clots are not as susceptible to the action of fibrinolytic enzymes and are virtually insoluble in denaturing solvents, such as 5M urea.
Factor XIIIa is an atypical coagulation enzyme since it is not a serine protease but rather a cysteine-containing, transamidating enzyme which catalyses the reaction between the amino acid side chains of lysine and glutamine to form an amide link with the elimination of ammonia according to the following scheme; ##STR1## When fibrin is the substrate, R.sub.1 --CONH.sub.2 and R.sub.2 --NH.sub.2 are glutamine and lysine side chains respectively in different chains of the fibrin polypeptide.
Factor XIIIa can also catalyse the cross-linking of other proteins. For example Factor XIIIa is known to link .alpha..sub.2 antiplasmin to fibrin and increase resistance to fibrinolysis. Moreover it can cause cross-links between a range of disparate structural and contractile proteins such as collagen, laminin actin, myosin, thrombospondin, vinculin and vitronectin or the like. It is believed that this property is part of the wound healing process and may have a role in the pathology of a number of diseases of tissue remodelling.
It is therefore desirable to provide an inhibitor of transglutaminases which inhibitor could be used, for example, in the treatnment of various pathological or thromboembolic events. Inhibitors of translutaminases have been described previously and these generally fall into four main categories:
(a) immunoglobulins directed at the enzyme; PA1 (b) low molecular weight substrates that compete with the natural protein substrates; PA1 (c) reagents that react with the active site of the enzyme; and PA1 (d) peptide fragments of Factor XIII itself.
These inhibitors are not suitable, for use in, for example, pharmaceutical formulations. for a variety of reasons, as follows:
Naturally circulating transglutaminase inhibitors have been identified previously as immunoglobulins directed at the sub-units of the transglutaminase. Such inhibitors give rise to a haemorrhagic condition caused by reduction in circulating factor XIII. U.S. Pat. No. 5,470,957 discloses using such immunoglobulins therapeutically by raising monoclonal antibodies to the transglutaminase enzyme sub-units by known techniques. A disadvantage associated with such antibodies as transglutaminase inhibitors is that they have high molecular weights and it is typically necessary to produce chimeric human analogues of the immunoglobulins before they can be used, for example, therapeutically in man.
WO91/10427 discloses transglutaminase inhibitors that are amines which act by linking to glutamine residues in one substrate to prevent cross-linking to another substrate. Such inhibitors are not very potent because they need to be present at the same concentrations as, or higher concentrations than, the natural substrate in order to have any significant inhibiting effect. Therefore they are only effective at concentrations in the region of approximately 50 .mu.M and above.
WO92/13530 discloses using various transglutaminase inhibitors which rely on the activity of transglutaminase being largely dependent on a reactive sulfhydryl group. Therefore any reagent that alkylates or oxidises this sulfhydryl group should inhibit the activity of the transglutaminase. Such reagents are, however, very reactive and also very unstable and are therefore particularly unsuitable for use in, for example, pharmaceutical or therapeutic treatment.
Attempts to provide peptidic inhibitors which might be expected to be more specific and less toxic has so far resulted only in compounds of low potency. For example, such inhibitors are described in U.S. Pat. No. 5,328,898 and by Achyuthan K E, Slaughter T F, Santiago M A et al; in J. Biol. Chem. 268: pp. 21284-21292, 1993; "Factor XIIIa derived peptides inhibit transglutaminase activity: localisation of substrate recognition sites".
Therefore, it is the purpose of one aspect of the present invention to provide a potent inhibitor of transglutaminase enzymes and which inhibitor can be used in, for example, pharmaceutical or therapeutic use.